Purification and Kinetic Studies of Wheat Bran βAmylase Evaluation of Subsite Affinities

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Abstract
1. β-Amylase [α-1,4-glucan maltohydrolase, EC 3.2.1.2] from wheat bran was purified to a single protein component as determined by ultracentrifugal analysis and polyacrylamide gel disc electrophoresis. The enzyme preparation was proved to be free from a-amylase [EC 3.2.1.1] contamination, as judged from the relationship between the blue value and the reducing value. 2. The Michaelis constant Km and the molecular activity k0 were determined at 25°C and pH 5.20 for the β-amylase-catalyzed hydrolyses of G3, G7, and G760, where Gn denotes a linear substrate with degree of polymerization n. The values of k0/Km were determined for a series of linear substrates, G3-G7, G12,5 and G760 from measurements of the initial rate for each substrate at a substrate concentration sufficiently lower than its Km value. 3. From the dependence of k0/Km on n, the number of subsites of this enzyme was estimated to be five. According to the subsite theory (30), the values of k0/Km and Km were used to evaluate the subsite affinity Ai of each subsite, where i is the subsite number counting from the terminal subsite at which the nonreducing end glucose residue of a substrate is to be situated. The values of (A1+A2), A3, A4 and A5 were evaluated, and the lower limit of A1 and the upper limit of A2 were also estimated. The large positive values of A1 and A4, and the negative values of A2 and A3 are consistent with the experimental data that maltotetraose is the smallest good substrate and that maltose and phenyl α-maltoside are not hydrolyzed to any detectable extent.

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